LOCUS       sequence-132531-        2282 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expression of cytosolic APEX2 in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 2282)
  AUTHORS   Lam SS, Martell JD, Kamer KJ, Deerinck TJ, Ellisman MH, Mootha VK, 
            Ting AY
  TITLE     Directed evolution of APEX2 for electron microscopy and proximity 
            labeling.
  JOURNAL   Nat Methods. 2014 Nov 24. doi: 10.1038/nmeth.3179.
  PUBMED    25419960
REFERENCE   2  (bases 1 to 2282)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Methods. 2014 Nov 24. doi: 10.1038/nmeth.3179."
COMMENT     Sequence Label: pTRC-APEX2
FEATURES             Location/Qualifiers
     source          1..2282
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     promoter        join(632..637,638..654,655..661)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    669..685
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     674..696
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             712..729
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             730..1476
                     /codon_start=1
                     /product="soybean ascorbate peroxidase, engineered to serve
                     as a monomeric tag for electron microscopy and live-cell 
                     proteomics (Lam et al., 2015)"
                     /label=APEX2
                     /note="more active than the original APEX due to the A134P 
                     mutation"
                     /translation="GKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAGT
                     FDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAV
                     EVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGHT
                     IGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDK
                     YAADEDAFFADYAEAHQKLSELGFADA"
     primer_bind     complement(1543..1560)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1713..1799
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1891..1918
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        1937..2028
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2247..2266)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 aaccatgcaa atgctgaatg agggcatcgt tcccactgcg atgctggttg ccaacgatca
       61 gatggcgctg ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg gtgcggatat
      121 ctcggtagtg ggatacgacg ataccgaaga cagctcatgt tatatcccgc cgttaaccac
      181 catcaaacag gattttcgcc tgctggggca aaccagcgtg gaccgcttgc tgcaactctc
      241 tcagggccag gcggtgaagg gcaatcagct gttgcccgtc tcactggtga aaagaaaaac
      301 caccctggcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
      361 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtaa
      421 gttagcgcga attgatctgg tttgacagct tatcatcgac tgcacggtgc accaatgctt
      481 ctggcgtcag gcagccatcg gaagctgtgg tatggctgtg caggtcgtaa atcactgcat
      541 aattcgtgtc gctcaaggcg cactcccgtt ctggataatg ttttttgcgc cgacatcata
      601 acggttctgg caaatattct gaaatgagct gttgacaatt aatcatccgg ctcgtataat
      661 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agaccatggc acaccaccac
      721 caccaccacg gaaagtctta cccaactgtg agtgctgatt accaggacgc cgttgagaag
      781 gcgaagaaga agctcagagg cttcatcgct gagaagagat gcgctcctct aatgctccgt
      841 ttggcattcc actctgctgg aacctttgac aagggcacga agaccggtgg acccttcgga
      901 accatcaagc accctgccga actggctcac agcgctaaca acggtcttga catcgctgtt
      961 aggcttttgg agccactcaa ggcggagttc cctattttga gctacgccga tttctaccag
     1021 ttggctggcg ttgttgccgt tgaggtcacg ggtggaccta aggttccatt ccaccctgga
     1081 agagaggaca agcctgagcc accaccagag ggtcgcttgc ccgatcccac taagggttct
     1141 gaccatttga gagatgtgtt tggcaaagct atggggctta ctgaccaaga tatcgttgct
     1201 ctatctgggg gtcacactat tggagctgca cacaaggagc gttctggatt tgagggtccc
     1261 tggacctcta atcctcttat tttcgacaac tcatacttca cggagttgtt gagtggtgag
     1321 aaggaaggtc tccttcagct accttctgac aaggctcttt tgtctgaccc tgtattccgc
     1381 cctctcgttg acaaatatgc agcggacgaa gatgccttct ttgctgatta cgctgaggct
     1441 caccaaaagc tttccgagct tgggtttgct gatgcctaag atccgctaga gtcgacctgc
     1501 aggcaagctt ggctgttttg gcggatgaga gaagattttc agcctgatac agattaaatc
     1561 agaacgcaga agcggtctga taaaacagaa tttgcctggc ggcagtagcg cggtggtccc
     1621 acctgacccc atgccgaact cagaagtgaa acgccgtagc gccgatggta gtgtggggtc
     1681 tccccatgcg agagtaggga actgccaggc atcaaataaa acgaaaggct cagtcgaaag
     1741 actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc
     1801 cgccgggagc ggatttgaac gttgcgaagc aacggcccgg agggtggcgg gcaggacgcc
     1861 cgccataaac tgccaggcat caaattaagc agaaggccat cctgacggat ggcctttttg
     1921 cgtttctaca aactcttttg tttatttttc taaatacatt caaatatgta tccgctcatg
     1981 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     2041 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     2101 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     2161 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     2221 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tgttgacgcc
     2281 tg
//
